Antibodies are naturally occurring biological agents that play a critical role in defending the body from pathogens. Antibodies, which are also commonly referred to as immunoglobulins, contain four polypeptides: two longer polypeptides (“heavy chains”) that are identical to one another and two shorter polypeptides (“light chains”) that are identical to one another. The heavy chains are paired with the light chains by disulfide bonds, and the two heavy chains are similarly bound to one another to create a tetrameric structure. Moreover, the heavy and light chains each contain a variable domain and one or more constant regions: the heavy chain includes one variable domain (VH) followed by three constant regions (CH1, CH2, and CH3), and the light chain includes one variable domain (VL) followed by a single constant region (CL).
The variable domains of each pair of light and heavy chains form the site that comes into contact with an antigen. Both VH and VL have the same general structure, with four framework regions (FRs), whose sequences are relatively conserved, connected by three hypervariable or complementarity determining regions (CDRs) (see Kabat et al., In “Sequences of Proteins of Immunological Interest,” U.S. Department of Health and Human Services, 1983; see also Chothia et al., J. Mol. Biol. 196:901-917, 1987). The four framework regions largely adopt a β-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs of VH and VL are held in close proximity by the FRs, and amino acid residues within the CDRs bind the antigen. More detailed accounts of the structure of variable domains can be found in Poljak et al. (Proc. Natl. Acad. Sci. USA 70:3305-3310, 1973) Segal et al. (Proc. Natl. Acad. Sci. USA 71:4298-4302, 1974), and Marquart et al. (J. Mol. Biol., 141:369-391, 1980).
Researchers have modified antibodies in various ways in order to study their function or to improve their utility as therapeutic agents. In some of the earliest modifications, researchers used double-stranded DNA sequences to express the VH or VL domains, but none of the sequence of the constant region (see, e.g., EP-A-0 088 994; Schering Corporation). Other fragments and chimeric antibodies have also been made. One particular type of chimera, commonly referred to as a CDR-grafted antibody, includes sequences from two antibodies that differ in species of origin (e.g., murine CDRs have been used in place of the naturally occurring CDRs in otherwise human antibodies; see, e.g., U.S. Pat. No. 5,225,539). Researchers hoped that such antibodies would be no more foreign to the human body than a fully human antibody, However, the utility of such antibodies has been restricted, at least in some cases, by a reduction in the antibody's affinity for the antigen. In an attempt to improve affinity, some of the amino acids in the FRs of CDR-grafted antibodies have been changed from those of the acceptor molecule (e.g., a human antibody) to those of the antibody that donated the CDRs (e.g., those of a murine antibody; see, e.g., U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; and U.S. Pat. No. 6,180,370). However, such modifications often result in molecules with increased immunogenicity. There remains a need for antibodies and other binding molecules that do not provoke a strong immune response yet bind strongly to antigen and methods for making such binding molecules.